Diacylglycerol kinase isoforms epsilon and zeta and methods of use thereof

Abstract

Two novel diacylglycerol kinase (DGK) isoforms are disclosed. The cDNA of one DGK isoform, designated DGK epsilon , is about 2.6 kb in length. The DGK epsilon cDNA has an open reading frame encoding 567 amino acids and has a predicted molecular mass of 63 kDA. DAG kinase epsilon is highly selective for arachidonate-containing diacylglycerol (DAG) substrates. The cDNA of the second isoform, designated DGK zeta , is 3.5 kb in length. The DGK zeta cDNA contains a single large open reading frame encoding a 928-amino acid protein with a predicted molecular mass of 103.9 kDa. An alternatively spliced muscle specific species of DGK zeta , DGK zeta -2 is also disclosed and characterized. DGK zeta is localized to the nucleus. A lysine-rich region with homology to the MARCKS protein is shown to be necessary and sufficient to confer nuclear localization to DGK zeta via a protein kinase C phosphorylation.

What is claimed and desired to be secured by United States Letters Patent is: 
     
       1. An isolated and purified nucleic acid sequence whose complement hybridizes to SEQ ID NO: 1 under the conditions of 65° C. overnight in a solution comprising 5×Denhardt's, 0.5×SSC, 0.1% SDS, and 0.1 mg/ml denatured herring sperm DNA, followed by washing once in 2×SSC, 0.1% SDS at room temperature for 20 minutes, and once in 0. 1 X SSC, 0.1% SDS at room temperature for 20 minutes, wherein said nucleic acid sequence codes for a protein having diacylglycerol kinase enzymatic activity. 
     
     
       2. The nucleic acid sequence defined in claim 1 wherein said nucleic acid sequence is subcloned into a plasmid. 
     
     
       3. The nucleic acid sequence defined in claim 1 wherein said nucleic acid sequence is subcloned into a prokaryotic or eukaryotic expression vector. 
     
     
       4. The nucleic acid sequence defined in claim 1 wherein said nucleic acid sequence is stably or transiently incorporated into a prokaryotic or eukaryotic host cell. 
     
     
       5. An in vitro method of decreasing the intracellular levels of diacylglycerol and increasing intracellular levels of phosphatidic acid comprising introducing into a eukaryotic cell the nucleic acid sequence of claim 1. 
     
     
       6. An isolated and purified nucleic acid, said nucleic acid comprising nucleotides which code for the amino acid sequence of SEQ ID NO: 2. 
     
     
       7. An isolated and purified nucleic acid which codes for human diacylglycerol kinase ε, said nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1. 
     
     
       8. A recombinant vector comprising the nucleic acid molecule of claim 7. 
     
     
       9. The recombinant vector of claim 8, wherein said recombinant vector is a plasmid. 
     
     
       10. The recombinant vector of claim 8, wherein said recombinant vector is a prokaryotic or eukaryotic expression vector. 
     
     
       11. The recombinant vector of claim 8, wherein the nucleic acid molecule is operably linked to a heterologous promoter. 
     
     
       12. A host cell comprising the nucleic acid of claim 7. 
     
     
       13. The host cell of claim 12, wherein the host cell is a eukaryotic host cell. 
     
     
       14. The host cell of claim 12, wherein the host cell is a prokaryotic host cell. 
     
     
       15. An in vitro method of decreasing the intracellular levels of diacylglycerol and increasing intracellular levels phosphatidic acid comprising introducing into a eukaryotic cell a nucleic acid sequence, said nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1.